Improved detection of chromosomal abnormalities in CLL by G-band analysis David Bohanna West...

Improved detection of Improved detection of chromosomal abnormalities in chromosomal abnormalities in

CLL by G-band analysisCLL by G-band analysis

David BohannaDavid Bohanna

West Midlands Regional Genetics Laboratory

CLLCLL

Most frequent leukaemia in the Western Most frequent leukaemia in the Western world world

Characterised by accumulation of long lived Characterised by accumulation of long lived B-lymphocytes in the bloodB-lymphocytes in the blood

Most (~80%) patients diagnosed early Most (~80%) patients diagnosed early without symptoms without symptoms – Detected as a result of a routine blood testDetected as a result of a routine blood test– Require markers that can predict the prognosis in these patientsRequire markers that can predict the prognosis in these patients


Prognostic MarkersPrognostic Markers

IGH mutational statusIGH mutational status– Mutated have superior survivalMutated have superior survival– High expense and expertiseHigh expense and expertise

CD38 and ZAP70 expressionCD38 and ZAP70 expression– Increased expression correlates with unmutated IGH (although Increased expression correlates with unmutated IGH (although

not perfectly)not perfectly)

CytogeneticsCytogenetics– G-band analysis (hampered by low MI)G-band analysis (hampered by low MI)– Prognostic information obtained from interphase FISH studiesPrognostic information obtained from interphase FISH studies


Interphase FISH prognostic markersInterphase FISH prognostic markers

PoorPoor: 17p (TP53) deletion, 11q (ATM) : 17p (TP53) deletion, 11q (ATM) deletiondeletion

IntermediateIntermediate: trisomy 12: trisomy 12

GoodGood: 13q mono allelic deletion*: 13q mono allelic deletion*

* Prognostic significance of bi allelic deletions of 13q uncertain


Byrd et al, 2004

13q14.3

13q34

Techniques at the WMRGLTechniques at the WMRGL

StandardStandard– 1. 1. Interphase FISH studiesInterphase FISH studies

Mainly TP53 and ATMMainly TP53 and ATMNot a whole genome screen like G-band analysisNot a whole genome screen like G-band analysis

– 2. Set up B-cell mitogen (TPA) stimulated 2. Set up B-cell mitogen (TPA) stimulated culturescultures

~40-50% abnormality rate~40-50% abnormality rate

ExperimentalExperimental– Use of alternative B-cell mitogensUse of alternative B-cell mitogens


Alternative B-cell mitogenAlternative B-cell mitogen

DNA fractions from DNA fractions from Mycobacterium Mycobacterium bovis bovis shown to have mitogenic effect shown to have mitogenic effect for B-cells in mice (Tokunaga et al, for B-cells in mice (Tokunaga et al, 1984)1984)

Synthetic CpG oligonucleotides (ODN) Synthetic CpG oligonucleotides (ODN) later shown to mimic these effectslater shown to mimic these effects

ODN that contain cytosine nucleotide ODN that contain cytosine nucleotide next to guanine nucleotide in the linear next to guanine nucleotide in the linear DNA sequenceDNA sequence

Mycobacterium bovis

West Midlands Regional Genetics LaboratoryCpG ODN

DSP30 and CLLDSP30 and CLL

DSP30 (CpG ODN)DSP30 (CpG ODN)– Used to stimulate B-cells in CLL by Claudia Haferlach’s group Used to stimulate B-cells in CLL by Claudia Haferlach’s group – Interleukin-2 (IL-2) requiredInterleukin-2 (IL-2) required– 72hr ONC (overnight colcemid) culture72hr ONC (overnight colcemid) culture

Haferlach et al (2007)Haferlach et al (2007)– 415/500 (83%) showed abnormalities visible by G-band analysis.415/500 (83%) showed abnormalities visible by G-band analysis.– 392/500 (78%) were abnormal by interphase FISH (with an 8 392/500 (78%) were abnormal by interphase FISH (with an 8

probe panel).probe panel).– 38% showed abnormalities by G-banding in addition to those 38% showed abnormalities by G-banding in addition to those

covered by the FISH panel (including complex karyotypes). covered by the FISH panel (including complex karyotypes).


AimsAims

Improve the G-band abnormality rate Improve the G-band abnormality rate achieved by WMRGLachieved by WMRGL

Compare the G-band abnormality rate of Compare the G-band abnormality rate of DSP30/IL-2 cultures with TPA culturesDSP30/IL-2 cultures with TPA cultures

Compare G-band abnormality rate of Compare G-band abnormality rate of DSP30/IL-2 cultures with interphase FISHDSP30/IL-2 cultures with interphase FISH


Methodology Methodology

35 patients with confirmed diagnosis of CLL35 patients with confirmed diagnosis of CLL

72hr DSP30/IL-2 (ONC) culture72hr DSP30/IL-2 (ONC) culture

6 day TPA (1hr colcemid) culture6 day TPA (1hr colcemid) culture

Interphase FISH on unstimulated cultureInterphase FISH on unstimulated culture– 13q14.313q14.3– ATM (11q22.3)ATM (11q22.3)– TP53 (17p13.1)TP53 (17p13.1)– CEP 12CEP 12


Methodology Methodology

G-band analysis of 20 cells from each of G-band analysis of 20 cells from each of the stimulated culturesthe stimulated cultures

100 cells (approx.) scored by interphase 100 cells (approx.) scored by interphase FISHFISH


Results SummaryResults Summary

DSP30/IL-2 DSP30/IL-2 ONC CulturesONC Cultures TPA CulturesTPA Cultures

Interphase FISH Interphase FISH (ATM,13q,p53,+12)(ATM,13q,p53,+12)

Success RateSuccess Rate 94% (33/35)94% (33/35) 66% (23/35)66% (23/35) 100% (35/35)100% (35/35)

Abnormality Abnormality RateRate 67% (22/33)67% (22/33) 57% (13/23)57% (13/23) 74% (26/35)74% (26/35)

Abnormality Abnormality Rate Rate

(Including (Including fails)fails) 63% (22/35)63% (22/35) 37% (13/35)37% (13/35) 74% (26/35)74% (26/35)


Comparison of G-band analysis of Comparison of G-band analysis of DSP30/IL-2 cultures and TPA culturesDSP30/IL-2 cultures and TPA cultures

22 cases failed/normal in 6DTPA cultures 22 cases failed/normal in 6DTPA cultures

10/22 abnormal in the DSP30/IL-2 culture10/22 abnormal in the DSP30/IL-2 culture

13 cases failed/normal in DSP30/IL-2 13 cases failed/normal in DSP30/IL-2 cultures cultures

1/13 abnormal in the 6DTPA culture1/13 abnormal in the 6DTPA culture


Comparison of G-band analysis of Comparison of G-band analysis of DSP30/IL-2 cultures with DSP30/IL-2 cultures with

interphase FISHinterphase FISH

13 cases failed/normal in DSP30/IL-2 cultures 13 cases failed/normal in DSP30/IL-2 cultures 7/13 abnormal by interphase FISH7/13 abnormal by interphase FISH

9 cases normal by interphase FISH 9 cases normal by interphase FISH 4/9 abnormal in DSP30/IL-2 cultures4/9 abnormal in DSP30/IL-2 cultures

11/26 cases abnormal by interphase FISH show 11/26 cases abnormal by interphase FISH show additional abnormalities in DSP30/IL-2 culturesadditional abnormalities in DSP30/IL-2 cultures


• 8/9 +12 clones detected in DSP30/IL-2 cultures

• 4/4 11q (ATM) and 17p (TP53) deletions detected in DSP30/IL-2 cultures


• 8/16 13q deletions detected in DSP30/IL-2 cultures

Abnormalities detected by FISH and G-band analysis

0

2

4

6

8

10

12

14

16

18

del (13q) trisomy 12 del (11q) del (17p)

Abnormality

No

. o

f ca

ses

FISH

DSP30/IL-2

TPA

Deletions of 13q Deletions of 13q 10 mono allelic deletions detected by FISH10 mono allelic deletions detected by FISH

4/10 were visible on G-banding in DSP30/IL-2 cultures 4/10 were visible on G-banding in DSP30/IL-2 cultures – 3/4 had one additional abnormality (undetected by interphase 3/4 had one additional abnormality (undetected by interphase

FISH)FISH)? Different prognosis? Different prognosis

6/10 had an apparently normal karyotype6/10 had an apparently normal karyotype– are the deletions sub-microscopic or under-represented in are the deletions sub-microscopic or under-represented in

metaphases? *metaphases? *

0/10 cases were associated with loss of 11q (ATM) or 17p (TP53) 0/10 cases were associated with loss of 11q (ATM) or 17p (TP53)

* Metaphase FISH of DSP30/IL-2 cultures showed abnormal population to be significantly under represented or not present in the metaphases.


del(13)(q12q14)

Deletions of 13q Deletions of 13q

6 bi allelic deletions detected by FISH6 bi allelic deletions detected by FISH

4/6 visible as mono allelic deletions in DSP30/IL-2 cultures4/6 visible as mono allelic deletions in DSP30/IL-2 cultures– 3/4 had additional abnormalities (2 ‘complex’)3/4 had additional abnormalities (2 ‘complex’)

2/6 had an apparently normal karyotype2/6 had an apparently normal karyotype– are the deletions sub-microscopic or under-represented in are the deletions sub-microscopic or under-represented in

metaphases?*metaphases?*

3/6 were associated with either loss of 11q (ATM) or 17p (TP53)3/6 were associated with either loss of 11q (ATM) or 17p (TP53)

*Metaphase FISH of DSP30/IL-2 cultures showed abnormal population to be significantly under represented or not present in the metaphases.

del(13)(q12q14)

Trisomy 12Trisomy 12

8/9 trisomy 12 clones were detected by G-8/9 trisomy 12 clones were detected by G-band analysis of DSP30/IL-2 culturesband analysis of DSP30/IL-2 cultures

4/9 trisomy 12 clones had a similar del/add(14q)

Del/add (14q) not detected in any other abnormal karyotypes

Potential sub group of +12 patients with different prognosis?


Case 1 Case 4

Case 10 Case 29

11q and 17p deletions11q and 17p deletions

2/2 cases had loss of 11q (ATM) by G-band analysis 2/2 cases had loss of 11q (ATM) by G-band analysis of DSP30/IL-2 cultures.of DSP30/IL-2 cultures.

2/2 cases had loss of 17p (TP53) by G-band analysis 2/2 cases had loss of 17p (TP53) by G-band analysis of DSP30/IL-2 cultures.of DSP30/IL-2 cultures.

In 4/4 cases with a poor cytogenetic marker, the In 4/4 cases with a poor cytogenetic marker, the abnormality was visible on G-band analysis of abnormality was visible on G-band analysis of DSP30/IL-2 cultures.DSP30/IL-2 cultures.


del(11)(q14q23)

add (17)(p1)

Is a ‘complex’ karyotype in CLL Is a ‘complex’ karyotype in CLL associated with a poor prognosis?associated with a poor prognosis?

Haferlach et al defined a ‘complex’ karyotype as Haferlach et al defined a ‘complex’ karyotype as ≥3 chromosome abnormalities≥3 chromosome abnormalities– Correlation with unmutated IGHCorrelation with unmutated IGH

Poor prognosisPoor prognosis

3/35 cases in our study had a ‘complex’ 3/35 cases in our study had a ‘complex’ karyotype of karyotype of ≥≥3 abnormalities 3 abnormalities – 2 cases had an 11q (ATM) or 17p (TP53) deletion2 cases had an 11q (ATM) or 17p (TP53) deletion

Poor prognosis using interphase FISH dataPoor prognosis using interphase FISH data

– 1 case had +121 case had +12Intermediate prognosis using interphase FISH data; now Intermediate prognosis using interphase FISH data; now poor prognosis?poor prognosis?


ConclusionsConclusions

For the 35 cases studied, the overall abnormality For the 35 cases studied, the overall abnormality rate in DSP30/IL-2 stimulated cultures (63%) is rate in DSP30/IL-2 stimulated cultures (63%) is higher than in TPA stimulated cultures (37%).higher than in TPA stimulated cultures (37%).

Standard practice at WMRGL has been changed Standard practice at WMRGL has been changed to include a DSP30/IL-2 stimulated culture for all to include a DSP30/IL-2 stimulated culture for all CLL referrals.CLL referrals.

The abnormality rate for interphase FISH studies The abnormality rate for interphase FISH studies was 74%, which was higher than that obtained was 74%, which was higher than that obtained using DSP30/IL-2.using DSP30/IL-2.


ConclusionsConclusions

Additional abnormalities were seen using G-Additional abnormalities were seen using G-banding compared to interphase FISH. These banding compared to interphase FISH. These included:included:– del/add(14q) within trisomy 12 clonesdel/add(14q) within trisomy 12 clones– additional abnormalities with del(13q)additional abnormalities with del(13q)– ‘ ‘complex’ karyotypescomplex’ karyotypes

““Prospective clinical trials should evaluate Prospective clinical trials should evaluate the prognostic impact of newly available data the prognostic impact of newly available data from G-band analysis of CLL casesfrom G-band analysis of CLL cases””..


Haferlach et al, 2007

AcknowledgementsAcknowledgements

Susan Rose Susan Rose Project SupervisorProject Supervisor

Mike Griffiths Mike Griffiths Head of WMRGL Oncology SectionHead of WMRGL Oncology Section

Mary Strachan Mary Strachan WMRGL Oncology SectionWMRGL Oncology Section

WMRGL Oncology sectionWMRGL Oncology section

WMRGL FISH sectionWMRGL FISH section

Professor Paul Moss Professor Paul Moss University Hospital BirminghamUniversity Hospital Birmingham


Improved detection of chromosomal abnormalities in CLL by G-band analysis David Bohanna West...

Documents

Transcript of Improved detection of chromosomal abnormalities in CLL by G-band analysis David Bohanna West...